Accumulation of protein aggregates and ubiquitin (Ub) into cytoplasmic inclusion bodies (IB) is the single most definitive diagnostic neuropathological marker of neurodegenerative disease. Despite this universal diagnostic significance, the cell biological mechanisms underlying IB formation and, indeed, whether formation of these structures reflects a pathogenic or protective process, remains an unresolved mystery. The long-term objective of this research project is to elucidate the molecular mechanisms that underlie IB formation and Ub deposition and to develop an integrated understanding of how mammalian cells respond to the chronic expression of pathogenic, aggregation-prone proteins. The research supported by this project during the previous funding period exploited single-cell analysis of a cellular model of Huntington's disease (HD) to show that, although mature IB contain both aggregated huntingtin (htt) and Ub, the two proteins are recruited to IB with vastly different kinetics. These observations, together with emerging data from genetic models of neurodegenerative disease, suggest a model in which chronic expression of a folding-defective aggregation-prone protein like htt, burdens the cell's proteostasis capacity (ie, the cell's ability to maintain the correct dynamic equilibrium between protein folding and degradation) leading to a progressive diversion of normal proteins from productive folding to the ubiquitin proteasome system, ultimately overwhelming the cell's capacity to degrade proteins. The research in this proposal aims to rigorously testand refine this emerging model using state-of-the-art proteomic and bioimaging technologies. These studies will provide a comprehensive understanding of the dynamic interaction between protein aggregation and proteostasis and will illuminate one of the longest-standing controversies in neurodegenerative disease biology.